Does Maternal Perinatal Probiotic Supplementation Alter the Intestinal Microbiota of Mother and...

Copyright © ESPGHAN and NASPGHAN. All rights reserved.

JPGN Journal of Pediatric Gastroenterology and Nutrition Publish Ahead of Print

DOI : 10.1097/MPG.0000000000000781

Does maternal perinatal probiotic supplementation alter the intestinal microbiota of

mother and child? A randomized controlled trial.

Christian Kvikne Dotterud1, M.D.; Ekaterina Avershina2, M.Sc.; Monika Sekelja2, Ph.D;

Melanie Rae Simpson1, M.D; Knut Rudi2, Ph.D; Ola Storrø1, Ph.D; Roar Johnsen1,

Ph.D; Torbjørn Øien, Ph.D1

1Department of Public Health and General Practice, Norwegian University of Science and

Technology, Trondheim, Norway.

2 Department of Chemistry, Biotechnology and Food Science, University of Life Sciences,

Aas, Norway

Corresponding author:

Christian Kvikne Dotterud

Medisinsk teknisk forskningssenter (MTFS)

N-7489 Trondheim

Telephone number: +47 73 59 50 00

Fax number: +47 73 59 75 77

E-Mail: [email protected]


Acknowledgments:

The authors thank all children and their parents for cooperation in this study; project

assistants Guri Helmersen and Else Bartnes; nurse Kirsten Øyum; Dr Rakel Berg; Dr Anne

Rø; Dr Marit Saunes; and all seven midwives in Trondheim.

Declaration of all sources of funding:

Supported by the Norwegian University of Science and Technology, the Norwegian

Research Council, Tine BA, Nidarosfondet, and Siemens AG. Research Manager Stein-

Erik Birkeland and Senior Researcher Anette Roll Mosland, Tine BA, contributed to the

practical accomplishment of the study. Tine BA had no role in the study design, data

collection, data analysis, interpretation of the study results, or writing of the manuscript.

Disclosure of potential conflict of interest:

All authors declare that they have no conflict of interest.

Word count: 2973

Number of figures: 5

Number of tables: 3

Supplemental Digital Content: 4

The trial was registered in ClinicalTrials.gov (identifier NCT00159523).

Supplemental digital content is available for this article. Direct URL citations appear in the

printed text, and links to the digital files are provided in the HTML text of this article on

the journal’s Web site (www.jpgn.org).


Abstract:

Objectives: Maternal probiotic supplementation has been shown to prevent the

development of atopic dermatitis (AD) in their offspring. We aimed to investigate whether

probiotics in pregnant and breastfeeding mothers altered the colonization pattern and the

diversity of the mothers’ and children’s intestinal microbiota.

Methods: In a randomized, double-blind trial, women received probiotic milk or placebo

from 36 weeks of gestation until 3 months postnatally whilst breastfeeding. The probiotic

milk contained Lactobacillus rhamnosus GG (LGG), L. acidophilus La-5 and

Bifidobacterium animalis subsp. lactis Bb-12. Stool samples were collected from the

mothers at 30 to 36 weeks gestation and 3 months after birth, and from the child at age 10

days, 3 months, 1 year and 2 years, and bacteria were analysed by quantitative PCR.

Additionally, stool samples from 3 months old and 2 years old children were characterized

using 16S rRNA gene deep sequencing to estimate the bacterial classes and genera, and the

alpha and beta diversity.

Results: Three months after birth, both the prevalence and the relative abundance of the

administered probiotic bacteria were significantly increased among the mothers in the

probiotic group compared to the placebo group. Only the LGG bacteria colonized the

children at 10 days and at 3 months of age. There were no significant differences in the

abundance of the administered probiotic bacteria between the groups at 1 and 2 years of

age. For the bacterial classes and genera, and alpha and beta diversity there were no

significant differences between the groups.


Conclusions: Different probiotic bacteria seem to have different ability to transfer from the

mother to the child. We found no evidence that the probiotics altered the microbial

composition or alpha and beta diversity of the children.

Keywords: probiotics, diversity, microbiota, infant


What is known

- A perinatal maternal probiotic supplementation has been shown to prevent the

development of atopic dermatitis in their offspring. The effect of such a

supplementation on the offspring´s intestinal microbiota is little studied.

What is new

- A perinatal supplement of three probiotic bacteria resulted in colonization of the

mothers.

- Only the LGG bacteria transiently colonized the children, indicating a differential

ability of probiotics to transfer from the mother to the child.

- There were no indications of the supplement to alter the microbial composition, or

alpha and beta diversity of the children.


Introduction

The revised hygiene hypothesis considers alterations in the intestinal microbiota during

infancy as an important contributor to the increase in the prevalence of atopic diseases (1).

Several trials have evaluated the role of probiotics in the prevention of atopic diseases and

atopic sensitization, and a meta-analysis provided evidence for a preventive effect on atopic

dermatitis (AD) in early life (2). In the Probiotics in the Prevention of Allergy among

Children in Trondheim (ProPACT), a randomized, double blind, placebo-controlled study,

we have previously reported a 40 % reduction in AD with maternal probiotic

supplementation (3).

The effect of maternal supplementation with probiotics on their children’s intestinal

microbiota is little studied. Maternal supplementation with Lactobacillus rhamnosus GG

(LGG) might lead to transient colonization of their infants (4) and has previously been

reported to affect Bifidobacterium colonization of the infant (5, 6). Recently, the role of

reduced diversity of the microbiota in atopic diseases and atopic sensitization has received

considerable scientific interest (7). Reduced diversity of the microbiota is associated with

an increased risk for developing atopic sensitization (8), IgE-associated AD (9, 10), AD

(11, 12), and asthma (13). The effect of probiotics on microbial diversity is not yet

conclusive, but a prenatal probiotic supplement with LGG has been reported not to increase

the diversity of the infants’ intestinal microbiota (14). Therefore, it is uncertain if the

preventive effect of maternal probiotic supplementation on AD is a result of intestinal

colonization with the probiotics among infants, an alteration of intestinal microbiota

diversity in the infants, or a combination of several mechanisms.


In this study we aimed to investigate whether a probiotic supplement, given to mothers

during the last four weeks of pregnancy and three months postnatally, altered the

colonization and the diversity of the mothers’ and children’s intestinal microbiota.

Materials and Methods

Participants and design

The design and the results of clinical outcomes in the ProPACT study are described in

detail elsewhere (3). Briefly, pregnant women were recruited during routine pregnancy

check-ups in primary health care in Trondheim, Norway, before week 36 of pregnancy.

Inclusion was not restricted to those with a family history of atopy.

The pregnant women were randomized without restrictions and with an allocation ratio of

1:1 to receive 250 ml probiotic low fat fermented milk or 250 ml placebo skimmed

fermented milk per day. Two hundred and fifty ml of probiotic milk, Biola®, contained 5 x

1010 colony-forming units of LGG, 5 x 1010 of Bifidobacterium animalis subsp. lactis Bb-

12 (Bb-12), and 5 x 109 of L. acidophilus La-5 (La-5) per day for the entire shelf life. The

placebo milk was sterile and contained no probiotic bacteria. The probiotic and placebo

products had equal taste, were produced, quality controlled, packed in neutral packages,

and distributed to the mothers by TINE BA, Oslo. LGG was quality controlled every

production day, and the BB-12 and La-5 were screened regularly throughout the study.

The study milk was given from 36 weeks gestation, and continued until the child was three

months old. The children were to be breastfed during this period.

Self-reported questionnaires on family history of atopy, gender, birth weight, breastfeeding,


prematurity, parity, maternal age, parental smoking behaviour, use of antibiotics and pet

exposure were collected among other data at baseline, and at 6 weeks, 1 year and 2 years of

age. Children were regarded as partially breastfed if they were breastfed 3 or more months,

and if breast milk substitute, different kinds of porridge, bread, vegetables, fruit,

commercially produced baby dinner, homemade baby dinner, fish, cows’ milk or eggs were

introduced before 3 months of age. Stool samples from pregnant women were collected at

inclusion during pregnancy week 30 to 36 and at 3 months after birth. Stool samples from

infants were collected from the diaper and transferred to a transport media by the parents

and stored at -18°C at home before transported to permanent storage at -80°C until

analysis. Four stool samples were collected from the child, at the age of 10 days, 3 months,

1 year and 2 years. All participating mother-infant pairs who submitted at least one stool

sample, and attended the 2 year clinical follow-up, were included in the current study.

The Regional Committee for Medical Research Ethics for Central Norway (Ref 097-03)

and the Norwegian Data Inspectorate approved the study (Ref 2003/953-3 KBE/-). One of

the parents signed a written informed consent formula.

Analysis of the microbiota

The protocol for the analysis of the microbiota was revised after the trial was commenced

due to methodological advances. Bacteria in the microbiota in the stool samples were

analysed by quantitative PCR (qPCR) using the primers and probes described in the online

content (http://links.lww.com/MPG/A442). We followed the protocol as previously

described (15). Briefly, the relative abundance of the bacteria were estimated by first

correcting for the differences in amplification efficiencies, and then normalizing the

detected quantities for each bacterial group relative to the total microbiota, as determined

by the Bacteria amplicon.


The microbial composition of stool samples from 3 month and 2 year old children was

characterized using 16S rRNA gene deep sequencing on Illumina MiSeq platform (Illumina

Inc., USA) (16). LGC magTM Nucleic extraction maxi kit (LGC Genomics, UK) was used

for DNA isolation on a KingFisher FLEX Magnetic Particle Processor (ThermoScientific,

USA) following manufacturer’s recommendations. 16S rRNA was amplified using

PRK341F and PRK806R primers adapted for the Illumina sequencing chemistry (Illumina

Inc., USA) (17). Sequencing with 250 bp chemistry was performed according to the

manufacturer’s recommendations at the Norwegian High-Throughput Sequencing Centre

(Oslo, Norway). The stool samples from 3 months and 2 years old children were analyzed

on separate Illumina chips. Resulting data were combined and analyzed using standard

workflow of QIIME pipeline (18). AmpliconNoise was used to denoise the data and

remove chimeras (19). Uclust was used for OTU picking (20). Sequences were clustered at

the 99 % similarity level, and the RDP classifier was used to assign taxonomic identity to

the resulting OTUs (21). In total were 7792425 sequences generated, with the median

number of reads 14132 and 293 samples with >4000 reads. Simpson’s index (1-D) and

Shannon’s index of diversity were used to assess alpha-diversity of the stool samples, while

UniFrac (weighted and unweighted) was used for beta-diversity analyses (22). Microbial

diversity and microbial composition analyses were based on 4000 randomly selected

sequences per sample. Those samples that failed to produce 4000 sequences were excluded

from further analyses.

Statistical analysis

The treatment allocation was conducted by the Department of Applied Clinical Research at

the Norwegian University of Science and Technology through a computer-generated

randomization list, and revealed to the researchers once all of the participants had

completed the endpoint examinations at 2 years of age. Researchers who analyzed the


microbiota were blinded to the group to which the participants were assigned.

Statistical analyses of the qPCR data were done using permutation analyses as previously

described.(15) Difference in alpha-diversity and bacterial composition at various time

points, was assessed by Kruskal-Wallis test. This is a non-parametric version of one-way

ANOVA for independent measurements (MATLAB® documentation, 2013). For beta-

diversity analyses, MANOVA were used to analyze the effect of age and probiotic

treatment on the principal coordinate scores from both weighted and unweighted Unifrac.

The null hypothesis was rejected at 5 % level. P-values were corrected for multiple testing

using Bonferroni. These adjusted p-values were reported only for those comparisons with

a statistically significant difference prior to the Bonferroni correction.

Results

Participants

From September 2003 to September 2005, 415 pregnant women were randomized to the

probiotic group or placebo group (Figure 1). At the clinical endpoint examination at 2 years

of age, 138 and 140 children in the probiotic and the placebo groups were assessed,

respectively. The compliance rates, defined as drinking ≥250 ml of study milk ≥50% of the

days, were 92.0% and 90.0% in the probiotic and placebo groups, respectively. The

numbers of faecal samples collected and analysed are illustrated in Figure 1. All

participating mother-infant pairs who attended the clinical examination at 2 years had

submitted at least one stool sample.

The study groups were comparable regarding baseline data and clinical characteristics

(Table 1).


Administered probiotic bacteria

At enrolment, the prevalence of pregnant women colonized with La-5 and Bb-12, as well as

the mean relative abundance of these bacteria in their stool samples, was similar in the

probiotic and placebo groups (Figure 2, Table 2). Three months after birth, both prevalence

and relative abundance of La-5 (p<0.005), LGG (p<0.005) and Bb-12 (p<0.005) was

significantly increased among the mothers in the probiotic group. The same effect was

observed for LGG in stool samples from infants at 10 days (p<0.005) and 3 months of age

(p<0.005), but the difference was no longer apparent at 1 year (p=0.783) or 2 years of age

(p=0.511) (Figure 2, Table 3). For La-5 and Bb-12, there were no statistically significant

differences between the groups at any time point in stool samples from the children.

Non-administered bacteria

There were no statistically significant differences between the groups at any time points in

the targeted qPCR analysis of non-administered bacteria (http://links.lww.com/MPG/A443).

The mean microbial composition of the stool samples from the infants at class level based

on 16s rRNA gene deep sequencing is illustrated in Figure 3. None of the bacterial classes

showed significant difference between the probiotic and the placebo groups at 3 months

and 2 years of age. No significant differences between the groups were found on the genus

level at 3 months and 2 years of age (data not shown).

Diversity of the microbiota

Alpha diversity was assessed by Simpson’s diversity index (1-D) and Shannon’s diversity

index based on 16s rRNA gene deep sequencing data (Figure 4). According to the

Simpson’s and Shannon’s index, there were no statistically significant differences between

the probiotic and placebo groups in the alpha diversity of the total microbiota in the stool


samples from infants at 3 months and at 2 years of age. Within each of the main phyla

Bacteroidetes, Firmicutes, Proteobacteria and Actinomycetes, neither Shannon’s, nor

Simpson’s indexes showed any statistically significant differences at any of the age

categories between the probiotic and the placebo groups (data not shown).

For the beta-diversity analyses, unweighted Unifrac showed the best separation based on

age, while there was no statistically significant difference between the probiotic and

placebo groups (Figure 5 and http://links.lww.com/MPG/A444). A separate analysis with

respect to probiotics for age split data revealed no significant difference between the groups

(p>0.9).

Discussion

In this study, the perinatal administration of the probiotic bacteria LGG, Bb-12 and La-5

lead to a significantly increased relative abundance of these bacteria in the mothers 3

months after birth. The abundance of the LGG bacteria was also increased in children from

the probiotic group compared with the placebo group at 10 days and 3 months of age.

However, at 1 and 2 years of age the difference between the groups was no longer apparent.

None of the other bacteria assessed with qPCR, or the main bacterial classes from the

Illumina sequencing analysis, were significantly different between the groups in either the

mothers or children. We found no indications that the administered probiotics alter the

microbial alpha or beta diversity of the children at 3 months and 2 years of age.

The strengths of this study are the randomized, controlled design, a long duration of

follow-up, and blinding of the intervention. Women were recruited from the general


population, and we believe the findings to be generalizable to pregnant women and children

under comparable conditions (3). The stool samples were collected from both the mothers

and the children at several time points, and the qPCR analysis of the microbiota provides

good sensitivity and specificity for the analysis of abundance of specific bacteria. The

diversity analysis was carried out using 16s rRNA gene deep sequencing which enables

deep sequencing characterization of the total microbial composition (16). Before correcting

for multiple testing, only the administrated probiotic bacteria showed statistical significant

differences between the probiotic and placebo groups. The results remained significant

after correction for multiple testing, ensuring a low chance of rejecting the null hypothesis

by chance. As none of the other comparisons showed a significant difference prior to

correction, the chance of failing to detect a true difference was minimized. The proportion

of stool samples successfully analysed was lower in the deep sequencing analysis

compared to the qPCR analysis. This was related to more stringent requirements in the

processing of the stool samples and the analyses, but considered independent of both the

treatment group assignment, and of the microbial composition. As such, the missing data

may be considered to be missing completely at random, without introduction of bias.

Participants for whom stool samples were included in the deep sequencing analysis were

comparable to the whole study population regarding baseline data. Mode of delivery may

modify the effect of probiotic supplement on the infant gut microbiota (23, 24), however

this information was not collected. According to the Medical Birth Registry of Norway, the

incidence of caesarian section in Trondheim varied between 14.1 to 15.4% during the study

period. We assume the incidence of caesarian section to be comparable in the present study

and equally distributed between the probiotic and placebo groups.

The evidence that oral ingestion of probiotic bacteria results in colonization is convincing,

and that this colonization is transient in children and lasts for approximately one to two


weeks after discontinuation of the administration (25, 26). In line with a small

observational study (27), and a study including a subsample of the original Kalliomäki

study (4), our study suggests that perinatal supplementation with LGG to mothers results in

a transient intestinal LGG colonization of the infants. Interestingly, in our study the

difference in colonization of the infants was only evident for LGG, not for La-5 and Bb-12.

This may indicate that the transfer of probiotic bacteria from the mothers to the infants is

strain specific. A previous study found that LGG administered during pregnancy increased

intestinal colonization of Bifidobacterium longum of the infant at 90 days of age, but

colonization of the infant with the administered LGG was not detected (5). Only one

previous study has reported the effect of probiotics on diversity. In this study, LGG was

administered during pregnancy only, and they did not find any effect on the infants’

microbial diversity (14). However, the sensitivity of the molecular technique used, T-

RFLP, is considered to be low (9). In the present study, using a more sensitive deep

sequencing technique and a larger number of participants, we did not find any evidence that

probiotics alter the children’s microbial diversity.

The mechanisms of transfer of the LGG strain from mother to child are not fully

understood. Traditionally it has been believed that the bacteria present in breast milk were

solely due to contamination from the mother’s skin or baby’s oral cavity (28). More

recently, it has been suggested that at least some of the bacteria present in the maternal gut

maybe transferred to the mammary glands via mononuclear immune cells in the systemic

circulation, and subsequently to the infant gut during breastfeeding (29). It has been

shown that maternal peripheral blood mononuclear cells and breast milk contain small

amounts of viable bacteria and a wide range of bacterial DNA signatures (30).


Furthermore, Lactobacillus reuteri has been detected in the colostrum after maternal

supplementation with the probiotic (31).

There are currently published results from 16 trials evaluating the preventive effect of

probiotics on AD, and a meta-analysis including 13 of these trials concluded that probiotics

are beneficial in the prevention of AD (2). In this meta-analysis, the sub-group analysis

suggests that pre- and postnatal administration may be required to achieve this preventative

effect. Subsequent to this meta-analysis, results from three trails have been published, all

with a pre- and postnatal administration (32-34). The mechanisms by which probiotics

exert this effect are uncertain. It was originally proposed that specific microbes in the gut

were important in prevention of atopic disease (35). These microbes were thought to exert a

preventative effect by antigenic competition, immune regulation, and stimulation of innate

immunity (36). However, the lack of clinical preventive effect in clinical trials with

postnatal supplementation alone suggests that the postnatal presence of these microbes may

not be sufficient to prevent AD, and the effect may partially occur during pregnancy or

postnatally via breast milk. A number of maternal cells have been found to cross the

placenta and induce an antigen-specific tolerance in the neonate (37), and it is suggested

that a probiotic supplement during pregnancy may modulate the fetal and placental immune

physiology in utero (38). However, currently there is no evidence from clinical trials to

support that a prenatal supplement alone is sufficient to prevent AD. It has also been

postulated that perinatal supplementation of probiotics might increase anti-inflammatory

immune regulatory factors in maternal breast milk which may lead to a preventative

mechanism that is independent of alterations in the intestinal microbiota of the infants (39,

40). The preventive effect found in trials with pre- and postnatal administration supports

the hypothesis that maternal supplementation contributes to the preventive effect. As

shown in the present study, some probiotic strains have the ability to transfer from the


mother to the child. This may help explain why the preventive effect is found both in trials

with maternal supplementation alone, and in trails were the supplement was administered

to the mothers prenatally and children postnatally.

In conclusion, the administered probiotics, LGG, Bb-12 and La-5, colonized the mothers,

but only the LGG bacteria colonized the children at 10 days and 3 months of age. This

indicates that different probiotic species have different ability to transfer from the mother to

the child. We found no indications of the administered probiotics to alter the microbial

composition, or alpha and beta diversity of the children at 3 months and 2 years of age.


References

1 Wold AE. The hygiene hypothesis revised: is the rising frequency of allergy due to

changes in the intestinal flora? Allergy 1998;53:20-5.

2 Pelucchi C, Chatenoud L, Turati F, et al. Probiotics supplementation during

pregnancy or infancy for the prevention of atopic dermatitis: a meta-analysis.

Epidemiology 2012;23:402-14.

3 Dotterud CK, Storro O, Johnsen R, et al. Probiotics in pregnant women to prevent

allergic disease: a randomized, double-blind trial. Br J Dermatol 2010;163:616-23.

4 Gueimonde M, Kalliomaki M, Isolauri E, et al. Probiotic intervention in neonates--

will permanent colonization ensue? J Pediatr Gastroenterol Nutr 2006;42:604-6.

5 Lahtinen SJ, Boyle RJ, Kivivuori S, et al. Prenatal probiotic administration can

influence Bifidobacterium microbiota development in infants at high risk of allergy. J

Allergy Clin Immunol 2009;123:499-501.

6 Gueimonde M, Sakata S, Kalliomaki M, et al. Effect of maternal consumption of

lactobacillus GG on transfer and establishment of fecal bifidobacterial microbiota in

neonates. J Pediatr Gastroenterol Nutr 2006;42:166-70.

7 Storro O, Avershina E, Rudi K. Diversity of intestinal microbiota in infancy and the

risk of allergic disease in childhood. Current opinion in allergy and clinical immunology

2013;13:257-62.

8 Bisgaard H, Li N, Bonnelykke K, et al. Reduced diversity of the intestinal

microbiota during infancy is associated with increased risk of allergic disease at school age.

J Allergy Clin Immunol 2011;128:646-52e1-5.

9 Abrahamsson TR, Jakobsson HE, Andersson AF, et al. Low diversity of the gut

microbiota in infants with atopic eczema. J Allergy Clin Immunol 2012;129:434-40,40 e1-

2.


10 Wang M, Karlsson C, Olsson C, et al. Reduced diversity in the early fecal

microbiota of infants with atopic eczema. J Allergy Clin Immunol 2008;121:129-34.

11 Forno E, Onderdonk AB, McCracken J, et al. Diversity of the gut microbiota and

eczema in early life. Clinical and molecular allergy : CMA 2008;6:11.

12 Ismail IH, Oppedisano F, Joseph SJ, et al. Reduced gut microbial diversity in early

life is associated with later development of eczema but not atopy in high-risk infants.

Pediatr Allergy Immunol 2012;23:674-81.

13 Abrahamsson TR, Jakobsson HE, Andersson AF, et al. Low gut microbiota

diversity in early infancy precedes asthma at school age. Clin Exp Allergy 2013;44:842-50.

14 Ismail IH, Oppedisano F, Joseph SJ, et al. Prenatal administration of Lactobacillus

rhamnosus has no effect on the diversity of the early infant gut microbiota. Pediatr Allergy

Immunol 2012;23:255-8.

15 Storro O, Oien T, Langsrud O, et al. Temporal variations in early gut microbial

colonization are associated with allergen-specific immunoglobulin E but not atopic eczema

at 2 years of age. Clin Exp Allergy 2011;41:1545-54.

16 Liu L, Li Y, Li S, et al. Comparison of next-generation sequencing systems. J

Biomed Biotechnol 2012;2012:251364.

17 Yu Y, Lee C, Kim J, et al. Group-specific primer and probe sets to detect

methanogenic communities using quantitative real-time polymerase chain reaction.

Biotechnology and bioengineering 2005;89:670-9.

18 Caporaso JG, Kuczynski J, Stombaugh J, et al. QIIME allows analysis of high-

throughput community sequencing data. Nature methods 2010;7:335-6.

19 Quince C, Lanzen A, Curtis TP, et al. Accurate determination of microbial diversity

from 454 pyrosequencing data. Nature methods 2009;6:639-41.

20 Edgar RC. Search and clustering orders of magnitude faster than BLAST.

Bioinformatics 2010;26:2460-1.


21 Wang Q, Garrity GM, Tiedje JM, et al. Naive Bayesian classifier for rapid

assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol

2007;73:5261-7.

22 Lozupone C, Knight R. UniFrac: a new phylogenetic method for comparing

microbial communities. Appl Environ Microbiol 2005;71:8228-35.

23 Makino H, Kushiro A, Ishikawa E, et al. Mother-to-infant transmission of intestinal

bifidobacterial strains has an impact on the early development of vaginally delivered

infant's microbiota. PloS one 2013;8:e78331.

24 Jakobsson HE, Abrahamsson TR, Jenmalm MC, et al. Decreased gut microbiota

diversity, delayed Bacteroidetes colonisation and reduced Th1 responses in infants

delivered by caesarean section. Gut 2014;63:559-66.

25 Alander M, Satokari R, Korpela R, et al. Persistence of colonization of human

colonic mucosa by a probiotic strain, Lactobacillus rhamnosus GG, after oral consumption.

Appl Environ Microbiol 1999;65:351-4.

26 Petschow BW, Figueroa R, Harris CL, et al. Effects of feeding an infant formula

containing Lactobacillus GG on the colonization of the intestine: a dose-response study in

healthy infants. J Clin Gastroenterol 2005;39:786-90.

27 Schultz M, Gottl C, Young RJ, et al. Administration of oral probiotic bacteria to

pregnant women causes temporary infantile colonization. J Pediatr Gastroenterol Nutr

2004;38:293-7.

28 West PA, Hewitt JH, Murphy OM. Influence of methods of collection and storage

on the bacteriology of human milk. The Journal of applied bacteriology 1979;46:269-77.

29 Fernandez L, Langa S, Martin V, et al. The human milk microbiota: origin and

potential roles in health and disease. Pharmacol Res 2013;69:1-10.

30 Perez PF, Dore J, Leclerc M, et al. Bacterial imprinting of the neonatal immune

system: lessons from maternal cells? Pediatrics 2007;119:e724-32.


31 Abrahamsson TR, Sinkiewicz G, Jakobsson T, et al. Probiotic lactobacilli in breast

milk and infant stool in relation to oral intake during the first year of life. J Pediatr

Gastroenterol Nutr 2009;49:349-54.

32 Rautava S, Kainonen E, Salminen S, et al. Maternal probiotic supplementation

during pregnancy and breast-feeding reduces the risk of eczema in the infant. J Allergy

Clin Immunol 2012;130:1355-60.

33 Ou CY, Kuo HC, Wang L, et al. Prenatal and postnatal probiotics reduces maternal

but not childhood allergic diseases: a randomized, double-blind, placebo-controlled trial.

Clin Exp Allergy 2012;42:1386-96.

34 Huurre A, Laitinen K, Rautava S, et al. Impact of maternal atopy and probiotic

supplementation during pregnancy on infant sensitization: a double-blind placebo-

controlled study. Clin Exp Allergy 2008;38:1342-8.

35 Kalliomaki M, Salminen S, Arvilommi H, et al. Probiotics in primary prevention of

atopic disease: a randomised placebo-controlled trial. Lancet 2001;357:1076-9.

36 Bach JF. The effect of infections on susceptibility to autoimmune and allergic

diseases. N Engl J Med 2002;347:911-20.

37 Mold JE, Michaelsson J, Burt TD, et al. Maternal alloantigens promote the

development of tolerogenic fetal regulatory T cells in utero. Science 2008;322:1562-5.

38 Rautava S, Collado MC, Salminen S, et al. Probiotics modulate host-microbe

interaction in the placenta and fetal gut: a randomized, double-blind, placebo-controlled

trial. Neonatology 2012;102:178-84.

39 Prescott SL, Wickens K, Westcott L, et al. Supplementation with Lactobacillus

rhamnosus or Bifidobacterium lactis probiotics in pregnancy increases cord blood

interferon-gamma and breast milk transforming growth factor-beta and immunoglobin A

detection. Clin Exp Allergy 2008;38:1606-14.


40 Rautava S, Kalliomaki M, Isolauri E. Probiotics during pregnancy and breast-

feeding might confer immunomodulatory protection against atopic disease in the infant. J

Allergy Clin Immunol 2002;109:119-21.


Figure 1. Flow of participants in the probiotic and placebo groups

a Number of collected stool samples

b Number of stool samples successfully analyzed.

Figure 2. Quantitative PCR analysis. Abundance of LGG relative to total bacterial load for

the mothers during pregnancy and 3 months postnatal, and the children at age 10 days, 3

months, 1 year and 2 years.

* p-value = 0.005

Figure 3. 16s rRNA gene deep sequencing analysis. Relative abundance of main bacteria at

class level in stool samples from probiotics and placebo group for children at age 3 months

and 2 years. The remainder to 100% represents low abundant or unidentified classes.

Figure 4. 16s rRNA gene deep sequencing analysis. Simpsons and Shannon’s alpha

diversity index of the total microbiota in the stool samples from probiotics and placebo

groups for children at 3 months and 2 years. Error bars represent 95% confidence interval

of the mean.

Figure 5. 16s rRNA gene deep sequencing analysis. Unweighted unifrac principal

coordinate plot of beta-diversity. The analyses were based on the genus level information.

PC 1 and 2 represent the first and second principal coordinate, explaining 30.9 and 14.1 %

of the variance, respectively.


qPCR analysis of microbiota

Mother pregnancy week 30-36 (na=128/nb=120)

Mother 3 months postnatal (na=130/ nb =124)

Child 10 days (na=138/ nb =133)

Child 3 months (na=128/ nb =121)

Child 1 year (na=127/ nb =119)

Child 2 years (na=126/ nb =121)

16s rRNA gene deep sequencing analysis of microbiota

Child 3 months (na=128/ nb =78)

Child 2 years (na=126/ nb =91)

qPCR analysis of microbiota

Mother pregnancy week 30-36 (na=127/nb=111)

Mother 3 months postnatal (n=122a/ nb =116)

Child 10 days (na =135/ nb =129)

Child 3 months (na =124/ nb =122)

Child 1 year (na =123/ nb =116)

Child 2 years (na =122/ nb =114)

16s rRNA gene deep sequencing analysis of microbiota

Child 3 months (na =124/ nb =65)

Child 2 years (na =122/ nb =88)

Discontinued intervention (n=53)

Child sick (n=1)

Declined further contact prenatally (n=25)

Declined further contact postnatally (n=27)

Lost to follow-up (n=11)

Moved (n=6)

Unable to contact (n=5)

Declined further contact (n=0)

Discontinued intervention (n=53)

Child sick (n=1)

Declined further contact prenatally (n=27)

Declined further contact postnatally (n=25)

Lost to follow-up (n=19)

Moved (n=9)

Unable to contact (n=5)

Declined further contact (n=5)

Allocated to probiotic group (n=211)

Received allocated intervention (n=210)

Allocated to placebo group (n=204)

Received allocated intervention (n=204)

Randomised

(n= 415)


Table 1. Baseline data and clinical characteristics (n=278)

Submitted at least one stool sample 16s rRNA gene deep sequencing analysis at 3 months of age

16s rRNA gene deep sequencing analysis at 2 years of age

Probiotic group (n=138)

Placebo group (n=140)





Mean (SD) Mean (SD) Mean (SD)

Age mother (years) 29.97 (3.84) 29.78 (4.10) 29.96 (3.98) 29.89 (4.27) 29.97 (3.97) 29.52 (4.22)

Education mother (years) 15.23 (2.02) 15.36 (2.08) 15.26 (2.13) 15.34 (2.14) 15.34 (1.94) 15.39 (2.02)

Education father (years) 14.87 (2.43) 14.59 (2.42) 14.98 (2.47) 14.38 (2.38) 14.81 (2.47) 14.62 (2.41)

Birth weight (grams) 3671 (484.0) 3595 (487.0) 3690 (430) 3555 (509) 3687 (505) 3625 (480)

n (%) n (%) n (%)

Gender (male) 72 (52.2) 57 (40.7) 35 (53.8) 28 (35.9) 48 (54.5) 41 (45.1)

Premature 14 (10.1) 8 (5.7) 5 (7.7) 7 (9.0) 8 (9.1) 3 (3.3)

Primiparous 74 (53.6) 92 (65.7) 33 (50.8) 53 (67.9) 47 (53.4) 59 (64.8)

Atopy in familyb 92 (66.7) 99 (71.2) 37 (56.9) 52 (67.5) 60 (68.2) 61 (67.8)

Smoking mother 3 (2.2) 4 (2.9) 1 (1.6) 2 (2.6) 3 (3.4) 3 (3.3)

Smoking father 9 (6.7) 19 (13.9) 4 (6.2) 9 (11.8) 7 (8.0) 13 (14.4)

Breast fed ≥ 3 months 134 (97.8) 138 (99.3) 62 (96.9) 76 (98.7) 86 (97.7) 90 (100)

Partially breastfedc 9 (9.0) 6 (6.5) 4 (8.7) 3 (6.0) 5 (7.9) 3 (4.8)

Pets at home 46 (33.6) 47 (33.6) 26 (40.0) 28 (37.2) 28 (32.2) 27 (29.7)

Child ever used antibiotics 57 (41.6) 59 (42.4) 28 (43.8) 39 (50.6) 40 (45.5) 35 (38.9)


a Submitted at least one stool sample.

b Mother, father or sibling ever had asthma or allergic rhinoconjunctivitis or AD.

c Information on introduction of breast milk substitute and the different food products before 3 months was obtained from the questionnaire collected at 1 year of age. In participants who submitted at least one stool sample, probiotic group n= 100 and placebo group n=93; analysed with 16s rRNA gene deep sequencing at 3 months of age, probiotic group n=46 and placebo group n= 50; and analysed with 16s rRNA gene deep sequencing at 2 years of age, probiotic group n=63 and placebo group n=62)


Table 2. Abundance of the administered probiotic bacteria for mothers during pregnancy

and 3 months postnatal, in the probiotic and placebo groups.

Pregnancy week 30-36 3 months postnatal na / nb

(%) meanc (SD)

p-valued na / nb (%)

mean (SD)

p-value

La-5 Probiotic group 9/111

(8.1) -5.47 (1.17) 0.319

70/116 (60.3)

-2.90 (2.49) 0.005e

Placebo group 4/120 (3.3)

-5.61 (0.87)

14/124 (11.3)

-5.15 (1.58)

LGG Probiotic group 6/111

(5.4) -3.53 (0.53) 0.388

74/116 (63.8)

-1.23 (1.97) 0.005e


-3.45 (0.74)

6/124 (4.8)

-3.32 (0.81)

Bb-12 Probiotic group 5/111

(4.5) -7.25 (0.60) 0.801

58/116 (50.0)

-5.31 (2.26) 0.005e


-7.27 (0.65)

5/124 (4.0)

-7.15 (1.01)

a Numbers of participants colonized b Number of stool samples successfully analyzed c The mean relative abundance compared to total bacterial load on a logarithmic scale. d p-value estimated from the mean relative abundance by permutation analyses e p-value corrected for multiple comparisons using Bonferroni correction.


Table 3. Abundance of administered probiotic bacteria for the infants at the age of 10 days, 3 months, 1 year and 2 years, in the probiotic and

placebo groups.

a Numbers of participants colonized

b Number of stool samples successfully analyzed c The mean relative abundance compared to total bacterial load on a logarithmic scale. d p-value estimated from the mean relative abundance by permutation analyses e Not applicable f p-value corrected for multiple comparisons using Bonferroni correction.

10 days 3 months 1 year 2 years na / nb

(%) meanc (SD)

p- valued na / nb (%)

mean (SD)

p-value na / nb (%)

mean (SD)

p-value na / nb (%)

mean (SD)

p-value

La-5

Probiotic group

1/129 (0.8) Nae

Na

2/122 (1.6) Na

Na

9/116 (7.8)

-5.40 (1.34) 0.650

31/114 (27.2)

-4.82 (1.70) 0.377 Placebo group

1/133 (0.8) Na 0/121

(0.0) Na 11/119 (9.2)

-5.47 (0.98)

36/121 (29.8)

-4.61 (1.93)

LGG

Probiotic group

50/129 (38.8)

-2.07 (1.84) 0.005f

56/122 (45.9)

-1.96 (1.75) 0.005f

12/116 (10.3)

-3.38 (0.79) 0.783

9/114 (7.9)

-3.45 (0.73) 0.511 Placebo group

7/133 (5.3)

-3.31 (0.76)

23/121 (19.0)

-2.89 (1.26)

12/119 (10.1)

-3.41 (0.77)

12/121 (9.9)

-3.38 (0.80)

Bb-12

Probiotic group

0/129 (0.0) Na

Na

4/122 (3.3) Na

Na

8/116 (6.9)

-7.18 (0.75) 0.671

11/114 (9.6)

-7.01 (1.22) 0.560 Placebo group

0/133 (0.0) Na 1/121

(0.8) Na 6/119 (5.0)

-7.22 (0.73)

15/121 (12.4)

-6.91 (1.28)

Does Maternal Perinatal Probiotic Supplementation Alter the Intestinal Microbiota of Mother and...

Documents

Transcript of Does Maternal Perinatal Probiotic Supplementation Alter the Intestinal Microbiota of Mother and...